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A PHYSIOLOGICAL STUDY OF TWO STRAINS 
OF FUSARIUM IN THEIR CAUSAL RE- 
LATION TO TUBER ROT AND WILT OF 
POTATO 



A DISSERTATION 

SUBMITTED TO THE FACULTY OF THE OGDEN GRADUATE SCHOOL 

OF SCIENCE IN CANDIDACY FOR THE DEGREE 

OF DOCTOR OF PHILOSOPHY 

(department of botany) 



BY 

GEORGE KONRAD KARL LINK 



A Private Edition 

Distributed by 

The University of Chicago Libraries 

Reprinted from 

The Botanical Gazette, Vol. LXII, No. 3 

Published by 

The University of Chicago Press 

Chicago, Illinois 



XTbe IHntversttp of Cbicago 



A PHYSIOLOGICAL STUDY OF TWO STRAINS 
OF FUSARIUM IN THEIR CAUSAL RE- 
LATION TO TUBER ROT AND WILT OF 
POTATO 



A DISSERTATION 

SUBMITTED TO THE FACULTY OP THE OGDEN GRADUATE SCHOOL 

OF SCIENCE IN CANDIDACY FOR THE DEGREE 

OF DOCTOR OF PHILOSOPHY 

(department of botany) 



BY 

GEORGE KONRAD KARL LINK 



A Private Edition 

Distributed by 

The University of Chicago Libraries 



Reprinted from 

The Botanical Gazette, Vol. LXII, No. 3 

Published by 

The University of Chicago Press 

Chicago, Illinois 



L»^»<^:i 



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KOy 27 '*** 



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VOLUME LXII NUMBER 3 



THE 

Botanical Gazette 

SEPTEMBER 1916 

a physiological study of two strains of 

fusarium in their causal relation to 

tuber rot and wilt of potato 

CONTRIBUTIONS FROM THE HULL BOTANICAL LABORATORY 2 1 9' 

George K. K. Link 

(with thirteen figures) 

There is little doubt among phytopathologists that members of 
the genus Fusarium play an important role in producing diseased 
conditions in many plants, both wild and cultivated. According to 
Wollenweber (41), Fusarium spp. produce wilt in members of 
the following families: Liliaceae, Bromeliaceae, Musaceae, Solana- 
ceae, Convolvulaceae, Leguminosae, Malvaceae, Linaceae, Cucur- 
bitaceae., Cruciferae, Compositae, Araliaceae, Caryophyllaceae, 
and Pedaliaceae. 

History 

The genus Fusarium was established by Link (20, 21) in 1809, 
and Fusarium species were reported on rotted and ring-discolored 
tubers by Martius in 1842, Hartig in 1846, and Schacht in 1856. 
Pizzigoni (29) and Wehmer (38, 39) demonstrated by experi- 
mental inoculation that Fusarium species can bring about tuber 
rot. They referred to the Fusarium in question as F. solani. 
Others, however, among them Frank (ii, 12) repeating their work, 
obtained negative results so far as Fusarium species were concerned ; 
while De Bary (6) and many others regarded the Fusarium spp. 
as nothing more than obligate saprophytes. 

' In cooperation with the Nebraska Agricultural Experiment Station. 

169 



I70 BOTANICAL GAZETTE [September 

The credit of first demonstrating experimentally the relation of 
Fusarium spp. to certain plant wilts belongs to Smith (34), who 
found a Fusarium responsible for watermelon wilt. Smith and 
Swingle (35) reported a potato wilt and tuber rot which they 
considered due to a Fusarium which they called F. oxysporum. 
They considered this organism identical with F. solani of Pizzigoni 
and Wehmer, and used the oldest name available, F. oxysporum 
(ScHLECHTENDAHL, 1824); howcver, they reported no experimental 
inoculations. Pethybridge and Bowers (28) reported a dry rot 
due to F. solani, and Longman (22) also reported a dry rot due 
to a Fusarium. 

Many pathologists and mycologists entertained considerable 
doubt as to the parasitic nature of Fusarium spp., while others were 
quite convinced of their parasitic nature. Sorauer (36) was quite 
positive in his decision, while Massee (25) wavered. Lindau (18) 
remained skeptical and referred to the F. oxysporum of Smith 
and Swingle as a "Mischart." Duggar (id) was quite positive 
in his decision. Much of this difference of opinion undoubtedly 
was due to the confusion that prevailed as to the status of F. solani, 
F. oxysporum, and the genus in general, since no basis for extended 
morphological study of the genus had been established, and even the 
genus itself had not been sharply defined. Massee (25) considered 
F. solani to be the conidial form of Nectria solani; while Reinke 
and Bertholdt (30) considered it the conidial form of Hypomyces 
solani. Lounsbury (23) tried to arbitrate the matter by suggest- 
ing that F. solani and F. oxysporum are one and the conidial stage 
of Nectria solani. Appel and Wollenweber (5) published a 
monograph in which they defined the genus and brought some 
order into the chaos of species. Among other radical changes they 
dropped F. oxysporum and established F. orthoceras in its place. 

Manns (24) demonstrated by experimental inoculation that a 
Fusarium, which he designated (following Smith and Swingle) as 
F. oxysporum, could produce tuber rot and wilt. He made no 
morphological studies, however, and undoubtedly had not had 
access to Appel and Wollenweber's monograph. 

Jamieson and Wollenweber (16) pubhshed an account of a 
dry rot of tubers induced by a Fusarium which they described as a 



I9i6] LINK—FUSARIUM 171 

new species {F. trichothecioides Wr.). They refer to it as "a wound 
parasite capable of destroying potato tubers " and say ''this disease 
is clearly differentiated from the wilt and dry rot ascribed by Smith 
and Swingle to F. oxysporum.'' 

Later, the writer (19) submitted his studies of a dry rot occurring 
among Nebraska potatoes as a thesis to the Graduate Faculty of 
the University of Nebraska. The work was done at the request 
of Dr. E. Mead Wilcox, and consisted in part of a study of the mor- 
phology of a Fusarium that had been isolated from dry rotted tubers 
in 1908 by Miss Venus W. Pool from potatoes that farmers had 
sent in from throughout the state during the season 1907-1908. 
Miss Pool established the causal relation of this Fusarium to the 
dry rot by experimental infection, and named the organism in 
manuscript F. pulverulentum, because of its powdery habit of 
growth. Both field and laboratory work were carried on for 
several years, and it was found that this organism caused primarily 
a dry rot of the tuber, and that it was not the F. oxysporum of Smith 
and Swingle, a culture of F. oxysporum having been furnished the 
laboratory for comparative work through the courtesy of Dr. 
Smith. The results were to have been published in 1911, and 
the organism was to be named F. pulverulentum, but upon the 
appearance of Appel and Wollenweber's monograph Dr. Wil- 
cox proposed to the writer that he reinvestigate the organism along 
the lines suggested by these authors. This was especially desirable 
since F. oxysporum had been dropped and several new species estab- 
lished. Not only was this carried out, but the whole etiology was 
gone over again and all of Miss Pool's results verified. It was 
found that Appel and Wollenweber (5) had not described the 
species, and consequently it was described as F. tuherivorum 
Wilcox and Link (40). It was so named because of the apparent 
restriction of its activity to tubers. 

A comparison of this paper and the paper of Jamieson and 
Wollenweber (16) made it seem quite likely that both were deal- 
ing with the same organism. The organism was isolated in the 
Washington laboratories from potatoes sent in from Washington, 
Nebraska, and other states in 19 10, and Wollenweber upon his 
arrival in the laboratory, using his monograph as the basis, described 



172 



BOTANICAL GAZETTE [September 



it as a new species. He told the writer in 1913 that he felt con- 
vinced that we had described one and the same thing. Comparative 
studies made by the writer during the past year verify this point of 
view, and since the Nebraska publication by Wilcox, Link, and 
Pool (40) did not appear in print until 1913, the name F. tricho- 
thecioides should be adopted. 

WoLLENWEBER (41, 42) published a further paper in which he 
categorized the Fusarium spp. very sharply, dividing the genus 
into sections on the basis of physiological (that is, pathogenicity) 
and morphological (that is, conidia and chlamydospores) characters. 
F. oxysporum was again estabhshed and taken as the representative 
of the section Elegans, which comprises vascular parasites; and 
F. trichothecioides was put into the section Discolor, which com- 
prises parench>Tna destroyers. He distinguished sharply between 
these and also between the vascular ring-discoloring Fusarium 
species of section Elegans and the tuber-rotting Fusarium species 
of sections Discolor, Gibbosum, Martiella, etc. 

Referring to the papers by Smith and Swingle (35), Manns 
(24), and others, particularly to that by Manns, he writes: "They 
do not separate fusarioses causing tuber rot from those causing both 
the wilt diseases of the plant and ring discoloration of the tuber, 
so that the reader might conclude that both wilt disease and tuber 
rot are caused by the same organism." Referring to his own experi- 
ments, he writes: "It also brings out the striking fact that the 
fungus, a typical xylem inhabitant, does not entirely destroy the 
tuber without the help of tuber rot Fusarium or bacteria," and 
" the fact that F. oxysporum causes the wilt of growing potato plants 
and only uses the xylem of the stem end of tubers for over- wintering, 
without producing a rot of the parenchyma, leads to interesting 
comparisons with the following 4 species which are able to destroy 
the tuber entirely from artificial wounds, namely, F. coeruleum 
(Lib.), F. trichothecioides Wr.," etc., and finally "the fact that the 
latter {F. oxysporum) cannot produce a tuber rot gives a biological 
contrast to the wound parasites of the tuber, and the fact that they 
cause the wilt disease of the growing plant presents a contrast to 
the saprophytes." 



I9i6] LINK—FUSARIUM 173 

Problem and method of attack 

In the spring and summer of 19 14 the writer discussed the 
Fusarium situation as outHned by Wollenweber with Dr. E. M. 
Wilcox and Dr. William Crocker. The former suggested that 
the whole situation ought to be gone over, and the latter that it 
would be of interest to search for the physiological basis of this 
alleged biological contrast. It is clear that, if the strict categories 
of Wollenweber exist, then potato parenchyma must possess 
either an absolute or an effective immunity toward Fusarium spp. 
of the Elegans section, and that Fusarium spp. of the Discolor 
section are either absolutely or practically unable to produce vascu- 
lar mycoses or wilts. 

The purpose of this research was twofold: (i) to determine 
whether such a sharp biological contrast exists; and (2) to deter- 
mine what is the physiological basis for such a contrast. Experi- 
mental infections of potato plants and tubers were used for the 
first phase of the problem. It was clear that the second phase might 
involve a great many considerations, such as the structural, com- 
positional, and metabolic nature of both host and parasite, as well 
as the relation of environmental factors to these. The important 
role played by the structural and compositional pecuHarities of the 
potato and the influence of external factors upon these is well 
illustrated by the studies of Appel and Kreitz (i, 3) on the efficacy 
of the cork layer in checking bacterial invasions of the tuber. 
Considerations of time and equipment limitations made it obliga- 
tory that the scope of the work be limited to a study of a few repre- 
sentative strains of the groups. 

The writer is under obligation to the Departments of Agricul- 
tural Chemistry, Horticulture, and Experimental Agronomy of the 
University of Nebraska Experiment Station for the use of materials 
and equipment; to Miss Ethel Beaty for help in much of the 
laborious routine; to Dr. Florence A. McCormick for valuable 
help in the anatomical and micro technical phases of the problem; 
and to Mr. R. A. Dawson for help in preparing the photographs. 

The writer decided to work with F. oxysporum as represent- 
ative of the vascular parasite section (Elegans), and with 



174 BOTANICAL GAZETTE [September 

F. trichothecioides or F. tuberivorum as representative of the 
parenchyma-invading section (Discolor). Since it was desirable 
that the identity of the organisms be well established, the writer 
asked Mr, W. A. Orton, in whose laboratories Dr. Wollenweber 
had carried out his recent work, for cultures of the organisms. It 
was impossible to get cultures which had been authenticated by Dr. 
Wollenweber, since he had gone to war, but through the courtesy 
of Mr. Orton, Mr. Carpenter (Dr. Wollenweber's assistant) 
furnished a strain of F. trichothecioides (no. 41, 1916) and a strain 
of F. oxysporum (no. 3345A). The other strains of F. trichotheci- 
oides used had been isolated by the writer in 191 1, and were 
described as F. tuherivoriim. Several strains of F. oxysporum 
isolated from Nebraska potatoes were also used. 

Pure cultures of these organisms were maintained on sterilized 
rice in plugged Erlenmeyer flasks, and these were used as a point 
of departure for all the work recorded. 

I. Infection experiments 

^ (i) experimental infection of tubers 

Tubers of the Early Ohio and Red Cobbler varieties were used 
in these experiments. Only sound tubers were selected, and these 
were thoroughly cleansed and sterilized before infection. At first 
they were sterilized by the formaldehyde gas method recommended 
by Wollenweber (41). Several difficulties were encountered in 
using this method. It was found very difficult to remove the last 
traces of the gas without contaminating the chamber, and the tubers 
often showed the characteristic formaldehyde vapor injuries that 
have been discussed in bulletins of the New York Experiment 
Station (13, 37). Consequently, the writer abandoned the first 
method and sterilized tubers by immersing in i : 1000 HgClj solu- 
tion for 1.5 hours. Inoculation was carried out by removing a 
piece of the cortex with a sterile cork borer, placing an infected 
grain of rice into the hole, and then replacing the piece of tuber 
tissue. The wound was then sealed with sterile grafting wax and 
the tuber placed into sterile chambers. This proved an efficient 
and convenient way of carrying out the great number of experi- 
mental inoculations made. 



I9i6] 



LINK—FUSARIUM i75 



The first inoculations were made in December 19 14. The cut 
ends of 20 Early Ohio tubers were wetted with spore suspensions 
of F. trichothecioides and 5 tubers were kept as controls. Four 
inoculated and one control potato were kept in each compartment 
at a temperature of 25° C. in an almost saturated atmosphere. 
After 4 weeks all of the inoculated tubers were in advanced stages 

of rot. 

On January 31, 3 potatoes were inoculated according to the 
second method with F. oxysporum, and 3 with F. trichothecioides, 
and kept at 20° C. until February 17. Two of the former set were 
slightly rotted and one totally, while the entire latter set was rotted 
severely. The controls showed no rot (fig. i). 

On January 15 another series was started which was kept at a 
temperature ranging from 15-20° C. until February 15. Six sets 
of 3 tubers each were started and each set was kept in a separate 
sterile chamber, 2 tubers of each set being inoculated by smearing 
cut surfaces with agar grown inoculum. Sets I, II, and III were 
inoculated with F. oxysporum, and sets IV, V, and VI with F. tri- 
chothecioides. In set I, one inoculated tuber was rotted, while the 
other and the control were sound; in set II, one was deeply rotted 
and the others sound; in set III, one was deeply rotted and the 
others sound; in set IV, two were rotted and the control sound; 
in set V, two tubers were rotted slightly and the control sound; and 
in set VI, two tubers were rotted and the control sound. 

F. oxysporum and F. trichothecioides were re-isolated from these 
rotted tubers by placing tissue cut from such tubers on plated glu- 
cose agar. Nothing other than the organism with which the tuber 
had been inoculated developed. Inoculum from these plates was 
used in infecting tubers again with the same results. 

Since these results were at variance with the statements of 
WOLLENWEBER the experiments were repeated with hundreds of 
tubers, and the results were verified. 

Discussion.— Tubers inoculated with F. oxysporum did not 
develop the ring discoloration that is considered characteristic of 
the activity of F. oxysporum, but a general rot of the whole tuber. 
Generally, however, this was not a dry rot, but a rot that resembles 
more the soft rots of bacterial origin, although it is not accompanied 



176 BOTANICAL GAZETTE [September 

by the offensive odors of bacterial rots, producing a blackening 
and softening of tissue which extends a considerable distance 
beyond the actual site of the organism. This was demonstrated 
microscopically and culturally. At times, however, especially 
in cold, dry conditions, a dry rot as typical as that produced by F. 
trichothecioides was produced. F. trichothecioides invariably pro- 
duced a dry rot with only a very limited darkened zone extending 
beyond the destroyed zone, made up of large cavities and a mixture 
of disintegrated, dry, shrivelled tissue and fungus tissue. No 
darkening extended beyond the actual site of the fungus and no 
softening of tissue occurred. Microscopic examination revealed 
the fact that F. trichothecioides attacked the tissue intracellularly 
and destroyed each cell completely before it proceeded to the 
neighboring cell, while F. oxysporum attacked the tissue intercellu- 
larly at first, and then attacked the cells intracellularly, but not 
until the tissue had been blackened and disorganized. In this way 
a softened tissue without cavities was produced. These rots pro- 
duced experimentally with pure cultures of F. oxysporum lend sup- 
port to the observations and conclusions of Smith and Swingle 
(35), Manns (24), who reports the occasional appearance of black 
specks in the parenchyma of tubers infected with F. oxysporum, and 
Jones (17), who attributes stem end rot of tubers to the activity of 
this organism, although they may have dealt with "Mischarten." 
Sherbakoff (33) reports certain strains of species of Elegans 
(using the section as a morphological group) to be tuber rotters. 
He distinguishes between Fusarium spp. that are tuber rotters and 
such as are vascular element inhabitants.' 

(2) experimental infection of living plants 
A series of experimental inoculations of healthy potato plants 
with F. oxysporum and F. trichothecioides were carried out, in an 
attempt to determine whether or not F. trichothecioides is unable to 

' After these experiments had been concluded and this paper written, a paper by 
Carpenter (7) has appeared. This represents a wholly independent although simul- 
taneously conducted piece of work. The results of Carpenter make it quite probable 
that the observations made by the writer on a few strains of F. oxysporutn are of 
quite general application, since he arrives at the same conclusions for numerous 
though different strains of F. oxysporum. His conclusions as to the method of attack 
by the fungus and the nature of the rot are practically identical with the writer's. 



I9i6] 



LINK—FUSARIUM 



177 



produce wilt, or whether the potato plant enjoys an effective or 
practical immunity rather than an absolute one. Even though 
WoLLENWEBER (41) did not consider F. trichothecioides a wilt 




^' 




B 



C 



Fig. I. — Tuber rot produced in laboratory with Fusarium oxysporutn, and F. tri- 
chothecioides; A, soft rot produced by F. o.vysporum, incubated at 20° C. for 17 days, 
Early Ohio variety; B, exterior of tuber rotted by F. trichothecioides, incubated at 20° C. 
for 17 days, Early Ohio variety; C, dry rot produced by F. trichothecioides, incubated 
at 20° C. for 17 days, Early Ohio variety. 



178 BOTANICAL GAZETTE [September 

producer in his 19 13 paper, there is a reference in the 19 12 paper by 
Jamieson and Wollenweber (16) to a wilt produced by F. tri- 
chothecioides . They referred to inoculation experiments, and 
report wilting in 12 days, ''accompanied by a yellowing of the 
leaves and a discoloration of the tissue." The results of all of the 
writer's attempts of 1911-1912 to produce wilting of potato plants 
with F. trichothecioides, excepting one, were negative. During the 
past winter, however, it was noticed again and again that sprouts 
of tubers experimentally infected with this organism were dying. 
Microscopic and cultural studies left no doubt that this organism 
was responsible for the death of the sprouts. 

Encouraged by these observations, the writer carried out some 
preliminary experiments on potato plants. Quartz was sterilized in 
6 inch flower pots in the autoclave, and 8 plants that were about 
10 cm. high were transplanted into these, the stems of some being 
smeared with rice infected with F. trichothecioides, and those of 
others with rice infected with F. oxysporum. The plants so inocu- 
lated and the controls were kept under bell jars. In three days the 
three plants smeared with F. oxysporum and two smeared with F. 
trichothecioides were dead, while the third one of the latter set and 
the controls remained healthy. The experiment was also conducted 
with potato plants growmg in the open bench in the greenhouse, with 
similar results. The soil in this case was not sterilized. 

The potato plants used in the following experiments were grown 
from sterilized tubers of the Early Ohio and Red Cobbler varieties 
in soil in 6 inch pots which had been thoroughly steriHzed by 
heating in an autoclave for 4 hours on two consecutive days at 
15 lb. pressure. The soil was watered with sterile water through- 
out the experiments. 

On February 15, fifteen pots were planted with Early Ohio 
tubers and the soil of one set of 5 was infected with rice infected 
with F. oxysporum, of another with rice infected with F. trichotheci- 
oides, while the third set was left as a control. The controls came 
up in due time, while not a single one of the others came up. This 
experiment was repeated several times, but in no case was so strik- 
ing a result obtained, although it often happened that some sprouts 
showed lesions, that some failed to come up, and that some were 



i9i6] LINK—FUSARIUM I'jg 

tardy in coming up. Fusarium spp. were isolated from such lesions. 
These lesions are identical in appearance with lesions found on 
potato stems and roots in the field which often are designated as 
*'foot disease" and ascribed to the activity of Rhhoctonia. Late 
in May other series were started and the soil was infected with rice 
and spore suspensions. No infections resulted at all, even though 
the inoculum was derived from the same source as that used in 
earHer experiments. 

On March 12, sprouts that were just breaking through the 
ground were uncovered and smeared with rice infected either with 
F. oxysporum or with F. trichothecioides , 6 sprouts being used in 
each set. The plants were wounded no more than was inevitable 
in removing the soil. The soil was then replaced. The soil in the 
controls was removed in the same way, but no inoculum was applied. 
The 12 sprouts to which inoculum had been appHed were killed, 
while the controls remained healthy. There was no spreading 
of the disease to other sprouts, even where an abundance of 
inoculum was applied. 

The affected sprouts reminded one forcibly of affected sprouts 
in potato fields in the spring. Here and there in the fields one 
finds sprouts that look sickly and small, which usually wilt and die 
or remain sickly and small. Upon examination of such sprouts, 
prominent brownish, watery lesions are found. At times such 
sprouts overcome the trouble and make a fair growth, at least 
until transpiration becomes excessive. These lesions also account 
for many of the "poor stands" or failures of potatoes to come 
up evenly. If one digs in where a sprout ought to have come 
up, one can often find a tuber that has sprouted, but whose sprouts 
have been cut off entirely by such lesions. Often lateral buds 
develop into branches on such decapitated sprouts, only to be cut 
off again. If such a tuber finally manages to get a shoot above the 
ground, the shoot is sickly and backward. In 191 2, 1913, and 1914 
the writer plated the inner tissue of many such sprouts and almost 
invariably obtained cultures of various Fusarium spp., although 
often associated with Rhhoctonia and bacteria. Infection experi- 
ments conducted with Rhizoctonia in 1912-1913 gave almost uni- 
formly negative results. The writer was at first inclined to refer 



i8o BOTANICAL GAZETTE [September 

the major part of the potato troubles to the activity of this organism. 
Even though it is not the sole or even the main cause of Nebraska 
potato troubles it may play an important role. The work of 
Appel (2), CoRSAULT (8), Drayton (9), and Morse and Schap- 
OVALOV (26) gave results similar to those obtained by Rolfs 
(3i»32). 




Fig. 2. — ^Wilt produced in laboratory with Fusariiim irichothecioides, and control 
plant; A, control, Early Ohio variety; B, wilting and drying of leaves, 4 days after 
inoculation. Early Ohio variety. 

On March 13, 24 plants grown in sterile soil were used in another 
experiment. These plants were about 10 cm. high at the time. 
The soil was removed from one shoot in each pot and the pots were 
arranged in 6 series. In series A the shoots were wounded and the 
wound smeared with F. oxysporum infected rice; in series B the 
sound stem was smeared with F. oxysporum infected rice; in series 
C the wounded shoots were smeared with F. trichothecioides infected 
rice; in series D the sound stems were smeared with F. tri- 
chothecioides infected rice; in series E no inoculum was applied to 
the wounded shoots; in series F the soil was merely removed and 
replaced (figs. 2 and 3). 



I9i6] 



LINK—FUSARIUM 



iSl 



On March 1 5 the following notes were taken. Series A : plant i , 
slight curling of leaves; 2, apparently sound; 3, curling of leaves; 
4, curling of leaves. Series B: plant i, drooping leaves; 2, lower 
leaves drooping, upper leaves drying; 3, apparently sound; 4, 
apparently sound. Series C: plant i, apparently sound; 2, some 
wilting; 3, some wilting; 4, some wilting. Series D: plants i, 2, 
and 3 apparently sound; 4, wilting. By March 21 the plants 




Fig. 



y^ B 

3. — Wilt produced in laboratory with Fusarimn oxysporum, and control 



plant; A, control, Early Ohio variety; B, wilting of lower leaves and curling of upper 
leaves, 4 days after inoculation, Early Ohio variety. 

infected with F. oxysporum showed a pronounced folding upward 
of leaves on the midrib, wilting and rolling on the margins of the 
leaves, the folding being most pronounced in the tips of the plants. 
The plants affected least showed discoloration on the margins, 
which at times was of a yellowish tint, at times purphsh to violet. 
The leaves of plants most severely affected showed a yellowing and 
burning of the leaf margins. One plant, inoculated with F. oxyspo- 
■rum, developed a pronounced rosette, but overcame this later, grow- 
ing into quite a normal plant (figs. 4 and 5). These symptoms 
remind one forcibly of certain symptoms of the leaf-roll disease which 
has received so much attention, and which has been made the subject 



l82 



BOTANICAL GAZETTE 



[SEPTEMBER 



of thorough study by Appel and his co-workers (2, 4). Eventually 
the plants infected with F. trichothecioides showed much severer 
s>Tiiptoms than those inoculated with F. oxysporum (fig. 6). Eight 
plants died in the former sets, and 3 in the latter. Plants infected 
\\dth F. trichothecioides showed such severe and rapid burning and 




k^..i£^l' 






Fig. 4. — Leaf roll and roselte ot field plant of the Pearl variety; August 19 12, 
at the U.S. Substation at Mitchell, Neb. 

drying up of leaves, that the typical wilting phenomena were 
scarcely realized. The vascular bundles were blackened and the 
blackening extended even into the petiole and the leaf veins. This 
rapid kilUng was at first strictly localized on that side of the plant 
to which the inoculum had been applied, even in the leaf, where the 
leaflets on one side of the midrib would be affected, and those on the 
other side not. Eventuallv in those cases in which killing of the 



I9i6] 



LINK—FUSARIUM 



183 



whole plant took place, the fungus girdled the whole stem, while 
plants that were not girdled lived on, even though one side 
was entirely destroyed. There was little lateral and subsequent 
vertical spreading of the fungus from one vascular strand to the 
other. These experiments were repeated with 25 other plants and 
in most cases the same symptoms were observed. These symptoms 
have been repeatedly observed in the dry land areas of Nebraska, 




J 



B 



Fig. 5. — Rosette produced in laboratory with Fusarium oxysporum, and control 
plant; A, control, Early Ohio variety; B, rosetted plant, 10 days after inoculation, 
Early Ohio variety. 



but have always been looked upon as cases of "sun scald," and 
and in previous experiments with wilting due to F. trichothecioides 
such cases were ignored. 

Plants grown in soil infected with F. oxysporum and F. tricho- 
thecioides showed severe lesions of root and stolons. Examination 
of roots affected with either organism showed that the cortical 
. regions are first and most severely attacked, not only intercellularly, 
but also intracellularly, the cells being packed full with hyphae. 
In most cases the cortex could be sloughed off with exceeding ease. 
From the cortex the organisms invaded the stelar regions, where 



1 84 



BOTANICAL GAZETTE 



[SEPTEMBER 



F. oxysporum makes greater headway than the other and there 
causes a vascular mycosis more frequently, which accounts for its 
designation as a vascular parasite (figs. 7 and 8). 

Discussion. — If plants, experimentally inoculated, showed 
only hght symptoms to begin with, most of them continued their 
growth with symptoms less severe than those shown in the field. 
If they showed severe symptoms early, these proved more severe and 




^ E 

Fig. 6. — Wilt and death of potato plants produced in laboratory with Fusarium 
trichothecioides, 12 days after inoculation; Early Ohio variety; wilting is restricted 
to the side to which inoculum was applied. 



more rapidly fatal than those in the field. The organisms in the 
field work much more insidiously, attacking the roots of the plant 
slowly but progressively, and permitting the plant, except in extreme 
cases, to readjust for its water requirements. These readjustments 
manifest themselves in the curling and rolling phenomena 
(figs. 4 and 9). 

Potato plants in the irrigated sections show this phenomenon 
nicely. As long as cultivation and irrigation are maintained, the 
plant develops new roots progressively higher up, and the infected 



I9i6] 



LINK—FUSARIUM 



185 



plants get along fairly well, showing slight curling and wilting, 
although tuber development occurs. When in midsummer 




Fig. 7.— Lesions on stem and roots produced in laboratory with Fiisarium oxy- 
sporum, 2 weeks after inoculation; Early Ohio variety. 



i86 



BOTANICAL GAZETTE 



[SEPTEMBER 




Fig. 8. — Root lesions produced in laboratory with Fusarium trichothecioides , curl- 
ing and rolling of leaves, 2 weeks after inoculation; Early Ohio variety. 



igi6] 



LINK—FUSARIUM 



187 



irrigation ceases and no more soil is heaped about the crown of the 
plant and transpiration requirements must be met by badly infected 




lower roots and a few healthy upper ones without the possibility 
of developing new roots, the plant soon succumbs. In this way we 
get the exceedingly frequent phenomenon of large plants, usually 



< 



Ph 



l88 BOTANICAL GAZETTE [September 

with many small tubers, wilting down suddenly after the last 
irrigation. The frequent occurrence of aerial tubers, the prevalence 
of excessive numbers of small tubers, and the occurrence of few 
abnormally large tubers on such plants is also attributable to the 
insidious manner of attack. The organisms attack the stolons and 
main stem as well as the roots. Stolons with tubers in all stages of 
maturity can be found partially or completely cut off by lesions. 
As the balance between the photosynthetic and storage centers in 
such plants is disturbed, new stolons are developed nearer and 
nearer the surface and the stolons that are not attacked develop 
abnormally large tubers. Often the plant responds to this disturb- 
ance in the assimilation-storage balance by producing swellings of 
the aerial parts of the plant, the so-called aerial tubers. Many 
large plants can be pulled up with ease, because lesions make separa- 
tion of the tops from the roots or even the basal portion of the stem 
easy. Such plants may show a comparatively sound main axis 

(fig- lo). 

Infection carried over by the mother tuber, which is frequent, 
rarely permits the growth of stems more than 20 cm. high, and 
seldom allows the development of tubers. An early attack from 
without upon the main stem leads to equally disastrous results. 

The wilts of the potato plant induced by Fusarium spp. have 
generally been considered vascular mycoses due to a clogging of the 
vascular elements. In fact, however, the symptoms are due to 
killing of the root system as much as to clogging of the vascular 
elements. It is true that members of the Elegans section, such 
as F. oxysporum, frequent the vascular elements, spreading in these 
rather than clogging them, but it is true also that they destroy roots 
in numbers. Again, even though some have referred to this disease 
as a root disease (Smith and Swingle 35), it is stated that the 
fungus enters a root, then spreads to the stelar part, and from there 
enters other roots and stolons. Just as much damage is done by the 
persistent attack from without upon roots and stolons, as noted 
by Manns (24). 

In the course of these experiments several questions were 
raised. The soil in these experiments surely was more severely 
infected with the organisms than soil under field conditions can 



I9i6] 



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189 



m^ 




100 BOTANICAL GAZETTE [September 

be, yet there were many plants grown in such soil that showed 
no infection whatsoever. Less success in producing wilt was 
observed as the season progressed. It remains a question whether 
this is due to a loss in vitality or to a gain in resistance in the plants, 
due to a change in the soil, tubers, or the organism. 

Whether the success in producing wilt with F. trichothecioides 
and the apparent waning of this power is due to a gain or regain of 
virulency and a subsequent loss again is also an unanswered question. 
Sherbakoff (33), working with Fusarium spp., got uniformly nega- 
tive results so far as producing wilt is concerned, and concluded 
that the results were due to a loss of virulency of the cultures or to 
some other important factor that had escaped attention. 

Summary 

It is quite apparent that some of the strains of F. oxysporum can 
cause tuber rot; that they can destroy tubers entirely without the 
aid of other Fusarium spp. or bacteria; that at least one Fusarium 
of the Discolor section (F. trichothecioides) can produce wilt of 
stem; and that the biological contrast drawn by Wollenweber 
between the Fusarium spp. is not as sharp as one would infer 
from his paper. It is possible that these strains of Fusarium spp. 
are morphologically identical with those described by Wollen- 
weber, but physiologically unlike them. That this rule, if it 
exists, is not so rigid generally, however, is noted by Sherbakoff 
(33); who found that no correlation exists between morphological 
relationship and pathogenicity. 

Although F. oxysporum is not absolutely unable to attack potato 
parenchyma, the potato tuber, in which usually only the xylem 
elements are invaded, enjoys an effective immunity from its attacks; 
and although F. trichothecioides can attack any subterranean part 
of the Hving potato plant, generally all parts excepting the mature 
tuber enjoy an effective immunity from its attacks. 

The data given in the second part of this paper may furnish a 
partial explanation of these phenomena. 



I9i6] LINK—FUSARIUM 191 

II. Ecology and physiology of the organisms 

METHOD AND DATA 

I. Temperature relations. — Observations of cultures grown 
at ordinary temperatures showed that there is a striking diiTerence 
in the rate of growth of the two organisms. Potato cylinder, rice, 
liquid potato, and glucose media, and glucose and potato agar cul- 
tures all showed that F. oxysporum makes a considerably greater 
initial growth at temperatures above 20° C. than does F. trichotheci- 
oides. At temperatures in the vicinity of 10-15° C, however, 
F. trichothecioides makes the greater initial growth, although these 
temperatures lie below its optimum. The same difference was noted 
in cultures on neutral and acid potato agar. This point was also 
tested with cultures on sterile slabs of potato tubers kept in Petri 
dishes. At 25° C. F. oxysporum covered such slabs completely 
when F. trichothecioides barely had made a start, while at 12° C. 
the situation was reversed. 

When I per cent liquid glucose media were inoculated with spore 
suspensions of F. oxysporum, visible growth was made in 16 hours; 
when F. trichothecioides was used, 30-42 hours elapsed before visible 
growth was made. This holds for temperatures above 20° C. The 
optimum temperature for F. oxysporum was about 30° C, and for 
F. trichothecioides about 20-22° C, both varying slightly with the 
medium used. The maximum for F. oxysporum lay between 38 and 
40° C. The optima and maxima were higher for cultures in potato 
extract than for glucose media cultures. The writer has not been 
able to determine the minima accurately because of inadequate 
apparatus. Humphrey (15) gives 4° C. as the minimum growth 
temperature for a certain strain of F. oxysporum. 

Potato agar cultures of F. oxysporum and F. trichothecioides 
could endure a temperature of 40° C. for 5 and for 20 hours respec- 
tively and remain viable. Exposure at 50° C. for 5 hours killed 
F. trichothecioides, but not F. oxysporum; while exposure for 20 
hours killed both. Some F. oxysporum cultures survived 5 hours 
exposure at 57° C. 

The growth relations were also checked up quantitatively. 
In these experiments, as well as in all the following ones, the method 



192 



BOTANICAL GAZETTE 



[SEPTEMBER 



suggested by Hasselbring (14) was followed. Erlenmeyer flasks 
of 200 cc. capacity were used with 50 cc. of solution per flask. The 
solutions in the flasks were autoclaved for 10 minutes at 7 lb. 
pressure, and then inoculated by means of sterile pipettes with a 
drop or two of spore suspension. The cultures were killed by 
adding 10 cc. of 10 per cent HCl to each flask. The cultures 
were then filtered off on tared Gooch crucibles prepared with 
asbestos, washed until acid free, and brought to constant weight in 
a Freas electric oven at 100° C, and the dry weight determined. 
It was found impossible at times to filter luxuriant cultures of 
F. oxysporum by this method, because of the tenacity with which 
this organism holds water. Consequently they were filtered on 
soft filter paper, transferred to tared Gooch crucibles, dried, and 
weighed. The other organism holds water with little tenacity and 
filters with ease. 

In all of experiments given below the following stock mineral 
solution was used: 20 gm. NH4NO3; 10 gm. KH2PO4; 5 gm. 
MgS04 per 1000 cc. H2O. When carbohydrates were employed, 

TABLE I 

Dry weight (in milligrams) after 20 days' growth in potato extract medium; 

ROOM temperature 





FUSARIUM OXYSPORUM 




Temperature 




35° 


30° 


25° 


12° 


I? II* 


—I? II* 


Flask I 


40 

47 
61 

49 


55 
78 
86 
73 


63 
68 
80 
70 


64 

66 

68 
66 


62 


86 






Flask 3 






Average 


62 


86 





FUSARIUM TRICHOTHECIOIDES 




Temperature 




35° 


30° 


25° 


12° 


i?ii* 


— i?ii* 


Flask I 















60 
64 
65 
63 


87 
100 

147 
III 


146 


83 


Flask 2 . . 




Flask 3 






Average 


146 


83 



*For 20 days (no growth), then at 25° C. for 25 days. 



i9i6] 



LINK—FUSARIUM 



193 



these were added at the rate of 10 gm. per Hter. Potato extract 
medium was made up by extracting 500 gm. ground potato tuber 
with 500 cc. H.O, and then adding 500 cc. of the foregomg solution 
to the extract. 

.A series of cultures (table II) was run at 12° C. and the amount 
of dry weight formed determined at 2 day intervals for 10 days. In 
this series the medium was at 12° C. at the time of inoculation. 

TABLE II 
Dry weight (in milligrams) in potato extract medium; temperature 12° C. 





FUSARIUM OXYSPORUM 


FUSARIUM TRICHOTHECIOIDES 




Number of days 


Number of days 




2 


4 


6 


8 


10 


2 


4 


6 


8 


10 


I 


0.2 
0.6 
0.4 


0.4 
0.4 
0.4 


5-2 

5-6 
5-4 




13.2 
13.0 
131 


1-4 
1.8 
1.6 


4.0 

4-4 
4.2 


9-4 
12.2 
10.8 




27.0 


II 

Average. 


44.6 
35-8 



Table III shows the growth by day intervals made for 10 days 
when levulose was used as the carbon source. The solutions 
were at the temperatures indicated at the time of inoculation. 

TABLE III 

Dry weight (in milligrams) formed by day intervals 





FUSARIUM TRICHOTHECIOIDES AT 2S°C. 




Number of days 




I 


2 


3 


4 


s 


6 


7 


8 


9 


10 


I 


0.2 
0.2 
0.2 


0.6 
2.4 


4.2 
18.0 
II. I 


12.8 

31.6 
22.2 


2Q.O 
324 
30-7 


19.2 

34-2 
26.7 


254 
27.2 
26.3 


19.4 
41.9 
30.6 


34-0 
47.7 
40.8 


43-6 


II 

Average . 


SO. 8 
47.2 











FUSARIUM OXYSPORUM AT 2S°C. 










Number of days 




I 


2 


3 


4 


S 


6 


7 


8 


9 


10 


I 


0.4 
0.8 
0.6 


1.8 
3-2 

2-5 


9.2 
10.8 
10. 


16.0 
29.2 
22.6 


23.6 

33-4 
28. 5 


19.6 
21.2 
20.4 


24.0 
275 
257 


24.1 
26.0 
25.0 


30-9 
34-8 
32.8 


370 


II 

Average . 


38.8 
37-9 



194 



BOTANICAL GAZETTE 
TABLE III— Continued 



[SEPTEMBER 





FuSARrUM OXYSPORUU AT 30°C. 




Number of days 




I 


2 


3 


4 


S 


6 


7 


8 


Q 


10 


I 

II 

Average. 


1 .0 
8.9 
4-9 


3-6 

5-2 

4.4 


154 
17.6 

16.5 


17.8 
19.0 
18.4 


37-6 
40.2 
38.9 


45-8 
77.8 
61.8 


55-8 
62.6 
59-2 


66.0 
69.6 
67.8 


78.2 
78.6 
78.4 


62. 2 

66.6 
64.4 






FUSARIUM TRICHOTHECIOIDES AT 25''c. 




Number of days 




I 


2 


3 


4 


s 


6 


7 


8 


9 


10 


Average . . . 


4-4 


19 


67.4 










262 






















FUSARIUM OXYSPORUM AT 30°C. 




Number of days 




I 


2 


3 


^"•1 ^ 


6 


7 


8 


9 


10 


Average . . . 


II. 2 


48 


108.6 










240.2 



















These tables show a tendency of F. trichothecioides to make a 
greater initial growth at low temperatures. At higher tempera- 
tures, however, unless above the optimum of F. trichothecioides, 
F. oxysporum, even though it made the greater initial growth, was 
soon overtaken and passed by F. trichothecioides. This was espe- 
cially marked when dextrose and levulose were used as carbon 
source. This may be the result of a faster though more super- 
ficial feeding of F. oxysporum, which makes it unable to use ma- 
terials as thoroughly as the other organism. This phenomenon is 
hardly a case of more rapid intoxication on the part of F. oxysporum. 

The results obtained with artificial media were verified by 
infection experiments conducted with potato tubers kept at various 
temperatures. Tubers of the Red Cobbler variety were used. 
These were inoculated on April i, and examined on May 27 
(table IV). See figs. 11 and 12. 



I9i6] 



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195 



It should be noted here that F. trichothecioides when inoculated 
into a tuber can grow at 30° C, while it cannot do so in artificial 
media; and that F. oxysporum can survive a temperature of 1° C. 
in artificial media, but not in the tuber. 

TABLE IV 
Condition of tubers at close of experiment 



Temperature 

30° c 

25° c 

12° C 

i°C 

1° C. for two weeks, then 
25° C. for two weeks . . 

-i°C 

— i°|C. for two weeks, then 
25° C. for two weeks. . 



Fusarium oxysporum 



Fusarium trichothecioides 



All completely rotted; 

sprouts killed 
All completely rotted; 

sprouts killed 
All with very slight rot 
No rot 

Slight rot in one tuber 
No rot 

No rot 



Slight rot in some 

All completely rotted; some 

sprouts killed 
AU completely rotted 
All with sUght rot 

All completely rotted 
No rot 

All completely rotted 



Discussion. — These results may, in part at least, explain why 
F. oxysporum, even though it can attack parenchyma and rot tubers, 
usually is not found in rotted tubers, while F. trichothecioides is. 
The ability of the latter to make a faster initial growth at the 
temperatures which prevail in the soil about digging time and in 
well kept storage places is probably the determining factor in this 
phenomenon. The experiments with tubers showed that F. tri- 
chothecioides made a great increase in growth rate when transferred 
from a low to a higher temperature. 

These temperature relations may also explain in part the fact 
that we usually find F. oxysporum producing wilt under field con- 
ditions, and lend support to the observations made by Orton (27), 
who reports potato wilt induced by Fusarium spp. to be pre- 
eminently a warm climate disease. F. trichothecioides can produce 
wilt, but the temperature conditions in the soil are such as to favor 
F. oxysporum, the maximum temperature of the former being 
the optimum of the latter. Humphrey (15), working in Washing- 
ton on the tomato wilt induced by F. oxysporum, came to the con- 
clusion that temperature differences in various parts of the state 
were determining factors for the appearance and non-appearance 
and severity of the disease. 



196 



BOTANICAL GAZETTE 



ISEPTEMBER 




^ 



c 




Fig. II. — Tuber rot of Red Cobbler variety produced by inoculation with Fusa- 
riitm oxysporum; A,B, external and sectional view of same tuber, incubated for 30 days 
12° C; C, D, external and sectional view of same tuber, incubated for 30 days at 25° C. 



I9i6] 



LINK—FUSARIUM 



197 




B 



D 



Fig. 12. — Tuber rot of Red Cobbler variety produced by inoculation with Fiisa- 
rium irichothecioides; A,B, external and sectional view of same tuber, incubated for 30 
days at 25° C; C, D, external and sectional view of same tuber, incubated for 30 days 
at 12° C. 



IqS botanical gazette [SEPTEMBER 

2. Growth habit. — It was observed in nearly all cultures that 
F. oxysporum not only made a greater initial growth at ordinary 
temperatures, but that it was at all temperatures much more of 
a surface grower than F. trichothecioides , making a superficial spread- 
ing growth, rather than the penetrating restricted intensive growth 
of the latter. Early sporulation was associated with the restricted 
growth habit of the latter (fig. 13). These habits were especially 
clearly marked on solid substrata, but even in liquid media F. oxy- 
sporum made a much less compact growth than the other species. 
It may be that the more spreading and extensive growth habit of 
F. oxysporum at all temperatures and its more rapid initial growth 
at temperatures above 10-15° C. are associated with a greater oxy- 
gen requirement than that possessed by F. trichothecioides. This 
would explain in part the frequenting of intercellular spaces and 
xylem elements by the former, and its consequent greater efficiency 
in causing vascular mycosis and wilt, as well as its tendency to cause 
bundle discoloration. The xylem elements of the stem end are 
undoubtedly infected while the tuber is yet in the soil, where 
temperature conditions are such as to favor the growth of F. oxy- 
sporum. Storage temperatures check the growth of this organism 
and the cells bordering the infected vascular elements shut the 
infected area off by suberizing their walls. Cultural experiments 
and microscopical studies show that cork is not absolutely impene- 
trable to these organisms, although it provides under normal con- 
ditions an effective barrier to the progress of both of these species. 
Because of the slower growth of F. trichothecioides at higher tempera- 
tures, the potato plant undoubtedly has a much better opportunity 
to guard itself by cork formation against this organism than against 
the other. 

3. The carbon sources of the two organisms. — ^A differ- 
ence in the metabolic requirements of two organisms, a difference 
in their ability to utilize various substances, or a difference in their 
abihty to tolerate the presence of substances may be factors of 
critical importance in determining which of the two will attack a 
given tissue or a given plant. These factors may determine also 
the modes of attack of an organism upon a tissue or a plant. Thus 
an organism that can digest pectinaceous material and not cellulose 



i9i6] 



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199 



would have to destroy a tissue whose walls are mainly cellulose 
by intercellular activity, while one that could digest cellulose might 



V£>^ 



^ B C 

Fig. 13. — Fusarium trichothecioides and F. oxysporum on sterile potato cylinders; 
A, C, cylinders inoculated with F. oxysporum, incubated for 2 days at 25° C; B, 
cylinder inoculated with F. trichothecioides, incubated for 2 days at 25° C. 

destroy this tissue by a primary cell invasion. Again, a greater 
abihty on the part of an organism to digest suberin, other things 



200 BOTANICAL GAZETTE [September 

being equal, would render it a much more formidable enemy of the 
potato plant than an organism without this ability, or possessing it 
to a less degree. This problem was attacked by making a study 
of the carbon sources of the organisms. The data reported here 
are only a beginning of this phase of the problem. 

Fifty cc. of nutrient solution were measured quantitatively into 
200 cc. Erlenmeyer flasks with a pipette. The flasks were then 
plugged with cotton, covered with tinfoil, and autoclaved. After 
cooling, 0.5 gm. of carbohydrate material was transferred quanti- 
tatively into each flask, and the flasks covered again with tinfoil 
and sterilized in a Freas oven by heating at 85° C. for one hour 
every 12 hours, for 6 consecutive days. The solutions were then 
incubated at 25° C. for 48 hours, so as to allow any contamina- 
tions present to appear. Low sterilization temperature was used to 
reduce hydrolysis of carbohydrates to a minimum. 

The dry weight determinations were made by the methods out- 
lined above. It was found advisable to kill two cultures of each 
set after 6 days, for the striking differences in rate of growth between 
the two organisms that were observed during the first 48-120 hours 
were obliterated by prolonged growth. The other 3 cultures were 
killed after 12 days' growth. The dry weight values do not show 
the differences in habit and rate of growth in the cultures as strik- 
ingly as they appeared to the eye. In many cases a visible growth 
was not determinable as dry weight. This is readily appreciated 
when we consider that moisture determinations indicated that the 
dry weight varied between 10 and 20 per cent of the wet weight. 

In the controls, consisting of the plain mineral medium without 
carbon material, F. oxysporum made a weighable growth in 12 days, 
though not in 6 days, while F. tricJiothecioides made no weighable 
growth even after 12 days. Another important observation was 
made. In no case was it necessary to reinoculate with F. oxysporum, 
while many F. tricJiothecioides inoculations failed. The latter 
undoubtedly is the slower starter and much more poorly equipped 
for sure and quick infection than the former. 

The figures in tables V-VIII represent milligrams of dry weight 
of material formed, except in those cases in which per cent is written. 
In such cases (cork, cellulose, and hemicellulose), the figures repre- 



I9i6] 



LINK—FUSARIUM 



20I 



sent the percentage of decrease in dry weight of material. The 
last weighing in these cases unavoidably included the dry weight 
of fungus material formed, so that the jfigures are higher than they 
ought to be. The differences in weight in these cases give only com- 
parative values of the amounts of material respired by the organ- 
isms. The filter paper used was the best Swedish paper, and the 
cork was obtained by skinning steamed potatoes, scrubbing the 
skin thoroughly, boiling it for 48 hours in distilled water, extract- 
ing for 48 hours in ether, and then boiling again with water. All 
figures represent averages, the composition of these figures being 
shown in tables VI and VII. In many cases there was a fair coin- 
cidence of the values, while in others a great disparity appeared. 
The averages probably would more nearly approximate the true 
value if a greater number of figures were available. 

TABLE V 

Dry weight (in milligrams) formed in 6 and i 2 days by Fusarium trichothecioides 

AND F. oxysporum 





Fusarium trichothecioides 


Fusarium oxysporum 




Number of days 


Number of days 




6 


12 


6 


12 


Ethyl alcohol 




1.2 
20.5 
136.3 
3S-0 
42.3 
52.0 

43-3 
81.0 
45-6 
61.0 
20.0 

47-3 

45 . per cent 

42 . 1 per cent 
33.4 per cent 

49 . per cent 

37-3 

123.0 

18.6 

14. 1 per cent 
7.3 per cent 
1 . per cent 
5 . 9 per cent 


I5-0 
17.0 

109.5 
78.7 
43-S 
49.0 
39.0 
74-5 

36. S 

35-S 
4.0 

44-5 

81.0 

54.3 per cent 

37.9 per cent 
110.5 

42.0 
102.0 

37.0 

27.1 per cent 

10. 1 per cent 
4. 2 5 per cent 

12.8 per cent 


18.5 

92.0 

112. 

43.0 
44.0 


Glycerine 

Mannit 

Arabinose 

Glucose 


12.0 
108.5 

S-2 
II-5 

2.0 
28.8 
56.5 

9-5 
130 

1-3 

12.4 


Mannose 


Galactose 

Fructose 


73.0 

77.6 

35.6 

SO. 3 

21.0 

54-6 
103.0 
127.0 

67 . 2 per cent 
210.3 

56.3 
90.0 

58.3 

25.7 

6 . 7 per cent 
6 . 7 per cent 
3.9 per cent 
o.S 


Saccharose 

Maltose 


Lactose 

Raffinose 


Potato starch 


Wheat starch 

Corn starch 

Soluble starch 

Dextrine 

Inulin 

Gum arable 

Gum tragacanth. . . 

Hemicellulose 

Cellulose 

Cork 

No carbon source. . 


22.6 per cent 

19 . 7 per cent 
45 . 2 per cent 

4-4 
63.6 

i-S 
67.5 per cent 
ID. I per cent 

4.0 per cent 
10. 1 per cent 











202 



BOTANICAL GAZETTE 



[SEPTEMBER 



Table V shows that quaHtatively the two organisms behave 
aHke in their ability to use all the carbon compounds tested. 
Quantitatively there is considerable difference, both as to rate of 
consumption and total growth after 12 days. F. oxysporum in 
general shows the greater speed of growth and greater growth after 
twelve days. In some cases F. trichothecioides shows the greater 
growth after 12 days. 

TABLE VI 

Dry \veight (in milligrams) formed when each flask received o . 5 gm. of 
mannit; concentration i per cent 



Number of flask 


FUSARIUM 


DXYSPORUM 


FUSARIUM TRICHOTHECIOIDES 


6 days 


12 days 


6 days 


12 days 


I 


93 
126 




107 
no 




II 






Ill 


108 

112 

116 

112 


130 

13s 
141 

136.3 


IV 






V 






Average 


109.5 


108.5 



TABLE VII 
Hemicellulose (in gm.) used in 12 days. 





FUSARIUM OXYSPORUM 


FUSARIUM TRICHOTHECIOIDES 


No. OF FLASK 


6 days 


6 days 




Grams of 
material 


Decrease in Percentage of 
weight decrease 


Grams of 
material 


Decrease in 
weight 


Percentage of 
decrease 


I 

II 


O.50SS 
0.5154 


0.045s 90 

0.0584 1 II. 3 

10 I 


0.5213 
. 4964 


0.0513 
0.0524 


9.8 

10.5 
10 I 


Average . 

















12 days 


12 days 


III 

IV 


0.5185 
0.5192 


0.0355 
0.0382 


6.1 
7.3 
6.7 


0.501 
0.5058 


0.028 
0.0488 


9.4 


Average . 


7.3 




' 











Table VIII gives the dry weight formed by the organisms when 
carbon acids were furnished as carbon sources. N/ioo solutions 
were used, excepting for asparagin and asparagenic acid, whose solu- 



i9i6] 



LTNK—FUSARIUM 



^03 



bility permitted only N/200 solutions. In the case of the higher 
fatty acids and oils, the material was weighed out as though N/ioo 
solutions were being prepared. With these no weighings of the 
material formed were attempted, but merely differences in luxuri- 
ance of growth were recorded. To those acids which showed no 
growth with N/ioo solutions, 5 cc. of 10 per cent glucose solution 
was added, making the sugar concentration i per cent, so as to 
determine whether the acid was merely non-usable, or whether 
it was toxic. Since it was found that some were toxic at N/ioo con- 
centration, lower -concentrations were made up also. The results 
are given in table IX. 



TABLE VIII 

Dry weight (m milligrams) formed with the following carbon compounds 
AS carbon sources 





FUSARIUM TRICHOTHECIOIDES 


FUSARIUM 


OXYSPORUM 




Number of days 


Number of days 




6 


12 


6 


12 


Formic acid N/ioo. . . . 


None 


None 


None 


None 


-I-5 cc. 10 per cent glu- 










cose solution 




" 




85.5 


Acetic acid N/ioo 


'' 


" 


1.2 


10.6 


-I-5 cc. 10 per cent glu- 










cose solution 


" 


106 


None 


None 


Proprionic acid N/ioo. . 


(1 


None 


(1 




-|-S cc. 10 per cent glu- 










cose solution 










Butyric acid N/ioo. .. . 


u 


" 


(1 


<( 


-\-S cc. 10 per cent glu- 










cose solution 


" 


■' 


" 




Glycocollic acid N/ioo. 


2-5 


7-3 


20.0 


8.6 


Lactic acid N/ioo 


0-3 


0.6 


8.0 


23.0 


Oxalic acid N/ioo 


Non-weighable 


Non-weighable 


Non-weighable 


Non-weighable 


Succinic acid N/ioo. . . 


Non-weighable 


Non-weighable 


8.5 


9-3 


Malic acid N/ioo 


Non-weighable 


Non-weighable 


4-5 


8.0 


Tartaric acid N/ioo. . . 


I.O 


0.93 


6.0 


4-3 


Citric acid N/ioo 


2.6 


6.6 


8.0 


6.6 


Aspartic acid N/200. . . 
Asparagin N/200 


I 6 




55 
55 


6.S 
5-3 


35 


3-8 


Tannic acid i per cent . 


1.0 


31.0 


0.2 


42.0 


Tannic acid . 5 per cent 


1.6 


61.0 


1.6 


41.0 


-|-5 cc. 10 per cent glu- 










cose 


3-5 


32.0 


■5.0 


47.0 


Control 0.5 gm. levu- 






lose 




82.0 




116. 











204 BOTANICAL GAZETTE 
TABLE Will— Continued 




1 


SEPTEMBER 






FuSARIUM 
TRICHOTHECIOIDES 


FUSARIUM 
OXYSPORUM 




12 days 


12 days 


Palmitic acid I 


No grc 
SUght 

u 

ExceUent 
SUght 
No 
No 

u 
u 

ExceUent 
No 

Poor 

ii 

a 

SUght 
No 

u 
u 
a 
u 

Fair 

(( 

u 
u 


wth 

(1 

(( 

u 

u 

u 
u 
u 
il 
u 
u 
it 
u 
u 
u 
u 
<( 
u 
u 
u 
u 
u 
u 
li 


Gooc 

u 
u 
11 
u 
u 
11 
a 
u 
u 
a 
» 

u 
u 
u 

No 

(( 

11 

u 
u 

Fair 

(( 

u 
(( 


i growth 

« 


« " II 


« III 

« IV 

+5 cc. 10 per cent dextrose, V 

Stearic acid, I 


<1 
(( 
u 
u 


« " II 


a 


" « II 


u 


" « III 


u 


" " IV 


u 


+ 5 cc. lo per cent dextrose, V 


u 


Oleic acid, I 


a 


« " II 


u 


« « III 


u 


« « IV 


u 


+S cc. lo per cent dextrose, V 


u 


Palm oil, I 


u 


" « II 


u 


« « III 


u 


« « IV 


a 


+5 cc. lo per cent dextrose, V 


u 


Olive oil, I 


u 


" "II 


u 


« « III 


u 


« "IV 


u 









A marked difference was found in the ability of the two organ- 
isms to use the fatty acids, F. trichothecioides being much more 
restricted in its ability. The experiments with alcohol and the 
acids also showed that the former organism was much more readily 
poisoned and inhibited in its growth. It was found that F. oxy- 
spormn grew well in i per cent ethyl alcohol, and that F. trichotheci- 
oides made no growth. The solution was then diluted one-half, 
whereupon F. trichothecioides made a good growth. This was 
clearly a case of inhibition. The growth of F. trichothecioides was 
inhibited by N/ioo acetic acid, as can be seen by the fact that it 
grew in N/125 concentration and that it grew in N/ioo when glu- 
cose was added, while F. oxysporum grew well in N/ioo acetic acid. 
N/ioo formic acid was toxic to F. trichothecioides, while it merely 
inhibited growth with the other organism. The latter grew in 
N/125 formic acid, while F. trichothecioides did not grow in N/500 



I9i6] 



LINK—FUSARIUM 



205 



solution. N/ioo proprionic acid was toxic to both, while both 
grew in N/250 solution. N/250 butyric acid was toxic to F. tri- 
chothecioides, while F. oxysporum grew in it. 

TABLE IX 

Dry weight (in milligrams) formed in 12 days in various concentrations 

OF ACID 





FUSARIUM 


OXYSPORUM 


FUSARIUM TRICHOTHECIOIDES 




N/iooo 


N/soo 


N/2SO 


N/I2S 


N/iooo 


N/soo 


N/2SO 


N/I2S 


Formic acid 
I 


Non- 

weigh- 

able 

Non- 

weigh- 

able 


Non- 

weigh- 

able 

Non- 

weigh- 

able 


Non- 

weigh- 

able 

Non- 

weigh- 

able 


Non- 

weigh- 

able 

Non- 

weigh- 

able 


Non- 

weigh- 

able 

Non- 

weigh- 

able 


None 


None 




II 


<£ 


Average .... 






















Acetic acid 
I 


1 






0.8 
0.8 
0.8 


1 .2 

2 

1.6 


3-2 

3-4 
iZ 


5.8 
6 6 


II 










Average .... 










6 2 














Proprionic acid 

I 

II 


1-4 
2.6 
2.0 


2.0 

2.4 
2.2 


50 

5.8 
5-4 


None 


1-4 
1.2 

1-3 


2.6 
3-4 
30 


4.0 
6.6 
5-3 


None 


Average.. . . 




Butyric acid 

I 

II 

Average. . . . 


4.8 
5-4 
51 


7.6 
9.0 


10.4 
13-8 
12. 1 


None 


2.4 
3-6 
30 


SO 
5.6 

S-3 


None 


None 








Oxalic acid 
I 


0.4 

0.8 
0.6 


0.4 

1.2 
0.8 


0.4 

1.2 
0.8 


1.2 

1.2 
1.2 


0.8 

0.8 
0.8 


Non- 
weigh- 

able 
Non- 
weigh- 

able 


Non- 
weigh- 

able 
Non- 
weigh- 

able 


Non- 

weigh- 

able 

0.2 

0. 1 


II 

Average .... 











A set of experiments was run also in which solanin in various 
percentages was added to glucose media. Because of the high 
cost of solanin, only i cc. solution was used in each test. The 
results are given in table X. 

These differences, that is, greater versatility in the use of carbon 
sources, greater resistance to inhibition, and intoxication, may well 



2o6 



BOTANICAL GAZETTE 



[SEPTEMBER 



play an important role in determining the difference in behavior 
of these two organisms. 

TABLE X 



Dry weight (in 


milligrams) formed by Fusarium 
F. trichothecioides m 6 days 


oxysporum 


AND 




Percentage solanin 




I 


2 


0,2S 


0. 226 





Fusarium oxysporum .... 
Fusarium trichothecioides 


I0.6 

2-5 


8.1 

2.4 


13.2 

7-8 


10 


13-8 
9-3 





Discussion. — -The versatiHty of these organisms in using various 
carbon sources in their metabohsm is of great interest. This almost 
omnivorous ability to use carbon compounds, including the simplest 
fatty acid, the highly oxidized fatty acids, the long carbon chain 
acids, the alcohols, mono- and poly-hybic, glycerin and fats, the 
mono-, di-, and poly-saccharides, including the dextrines, starches, 
hemicelluloses, and true celluloses, assigns to them an important 
role in the carbon cycle, and at the same time must help render 
them the formidable and destructive enemies of the root crops that 
they are. 

The methods suggested by Appel (2), namely, rigid inspection 
of potato fields, immediate destruction of all plants that show the 
slightest symptoms, quarantining of non-certified seed stock, alone 
give promise of keeping these troubles in check. Disinfection of 
storage cellars and of potatoes when put into storage, together with 
storage at proper temperature, will help combat these diseases, 
especially the dry rot induced by F. trichothecioides. 

Conclusions 

1. Fusarium tuberivorum Wilcox and Link is the same as Fusa- 
rium trichothecioides Woll. 

2. Both Fusarium oxysporum and F. trichothecioides can produce 
both tuber rot and wilt of the potato plant. 

3. The wilt is induced by destruction of the root system and 
by clogging of the xylem elements in the stem, and is, in mild 
cases, marked by such symptoms as discoloration of leaves, curling 
and rolling of leaves, and production of aerial tubers. 



i9i6] LINK—FUSARIUM 207 

4. Under field and storage conditions Fusarium oxysporum is 
more probably responsible for wilt than is F. trichothecioides, and 
the latter more responsible for tuber rotting. 

5. The optimum and maximum temperatures of Fusarium oxy- 
sporum are higher than those of F. trichothecioides. F. trichothe- 
cioides, however, grows well at 8-10° C, while F. oxysporum does 
not. These facts may explain in part the fact that F. oxysporum 
produces more wilt than F. trichothecioides, and that the latter 
causes more tuber rot. 

6. Fusarium oxysporum has a more rapid, superficial, and 
spreading habit of growth than has F. trichothecioides. This may 
be associated with a greater oxygen requirement for F. oxysporum, 
and may account for the frequenting of xylem elements by this 
fungus. 

7. Both organisms possess a striking abihty to use the most 
diverse carbon materials as carbon sources in their metabolism. 
Fusarium oxysporum has a greater range in its ability, and can 
utilize the materials more readily, although not so completely as 
does F. trichothecioides. 

8. Fusarium oxysporum is less subject to inhibition in growth 
and intoxication than is F. trichothecioides. 

9. Solanin is not toxic to either organism, although it seems to 
inhibit somewhat the growth of Fusarium trichothecioides. 

The writer acknowledges his indebtedness to Dr. E. Mead 
Wilcox and to Dr. William Crocker. They not only made this 
research possible, but they gave freely of advice and criticism, and 
lent encouragement by their interest in the progress of the investi- 
gation. 

Agricultural Experiment Station 
University of Nebraska 



LITERATURE CITED 

1. Appel, Otto, Untersuchungen iiber die Schwarzbeinigkeit und die durch 
Bakterien hervorgerufene KnoUenfaule der Kartoffel. Arb. Biol. Abth. 
Land- u. Forstw. Kais. Gesund. 3:362-432. pi. i. figs. 1-15. 1903. 

2. , Leaf roll diseases of the potato. Phytopathology 5:139-148. 1915- 



2o8 BOTANICAL GAZETTE [September 

3. Appel, O., and Kreitz, Wilhelm, Untersuchungen iiber die Schale ver- 
schiedener Kartoffelsorten und ihre Beeinflussung durch Bodenverhaltnisse, 
Feuchtigkeit und Diingung. Arb. Kais. Biol. Anst. Land- u. Forstw. 
6:1-27. pis. 2. figs. i-io. 1908. 

4. Appel, O., and Schlumberger, Otto, Die Blattroll Krankheit und unsere 
Kartoffelernten. Berlin, pp. 102. ^/5. j. 191 1. This contains a complete 
bibliography. 

5. Appel, O., and Wollenweber, H. W., Grundlagen einer Monographie der 
Gattung Fusarium (Link). Arb. Kais. Biol. Anst. Land- u. Forstw. 
8:1-207. P^^- 4- fis^- ^~^- 1910. 

6. Bary, a. De, Die gegenwartig-herrschende Kartoffelkrankheit, ihre Ur- 
sache, und ihre Verhiitung. Eine pflanzenphysiologische Untersuchung 
in allgemein verstandlicher Form dargestellt. Leipzig, pp. 755. 1861. 

7. Carpenter, C. W., Some potato tuber-rots caused by species of Fusarium. 
Jour. Agric. Research. Dept. Agric. 5: no. 15. 183-209. pis. 7. 191 5. 

8. CoRSAULT, J. H., Studies of the Rhizoctonia disease of potatoes. Phyto- 
pathology 5:293-294. 1915. 

9. Drayton, F. L., The Rhizoctonia lesions on potato stems. Phytopathology 
5:59-63. pi. I. fig. I. 1915. 

10. DuGGAR, B. M., Fungous diseases of plants, with chapters on physiology, 
culture methods, and technique. Boston. 1909. 

11. Frank, A. B., Die Krankheiten der Pflanzen. Breslau. 1896. 

12. , Untersuchungen iiber die verschiedenen Erreger der Kar toff elf aule. 

Ber. Deutsch. Bot. Gesells. 16:273-280. 1898. 

13. Gloyer, W. O., The efficiency of formaldehyde in the treatment of seed 
potatoes for Rhizoctonia. Bull. no. 370. N.Y. Exp. Sta. 417-431. 1913. 

14. Hasselbring, The carbon assimilation of Penicillium. Bot. Gaz. 45: 176- 
193. 1908. 

15. Humphrey, H. B., Studies on the relation of certain species of Fusarium 
to the tomato blight of the Pacific Northwest. Bull. State Coll. Wash. 
Agric. Exp. Sta. no. 115. pp. 21^ pis. 5. 1914. 

16. Jamieson, C. O., and Wollenweber, H. W., An external dry rot of potato 
tubers caused by Fusarium trichothecioides WoUenw. Jour. Wash; Acad. 
Sci. 3:146-152. _^g. I. 1912. 

17. Jones, L. R., Potato diseases in Wisconsin and their control. Circ. of 
Information. Wis. Exp. Sta. 36:1-10. 1912. 

18. LiNDAu, G., Die pf^anzlichen Parasiten. Sorauer's Hand. d. Pflanzenkr. 
2: pp. 550. figs. 62. 1908. 

19. Link, Geo. K. K., Studies of a Fusarium causing dry rot of Irish potatoes. 
Thesis, Univ. of Neb. May 191 2. 

20. Link, H. F., Observationes in ordines plantarum naturales. Mag. Gesells. 
Naturf. Freunde 2:3-42. 1809. 

21. , Species Hyphomyceten et Gymnocetum. Pars i. 1824. 



i9i6] LINK—FUSARIUM 



209 



22. Longman, Sibyl, The dry rot of potatoes. Jour. Linn. Soc. Bot. 39: 
120-129. P^- ^- 1909. 

23. LouNSBURY, C. P., Dry rot of the potato. Agric. Jour. Cape Good Hope 
35:42-48. figs. 31. 1909. 

24. Manns, Thos. F., The Fusarium blight and dry rot of the potato. Bull, 
no. 229. Ohio Exp. Sta. 299-336. pis. 1-15. 191 1. 

25. Massee, G., Textbook of plant diseases. London. 1904. 

26. Morse, W. J., and Schapovalov, M., The Rhizoctonia disease of potatoes. 
Bull. no. 230. Maine Exp. Sta. 1914. 

27. Orton, W. a., Potato wilt, leaf roll, and related diseases. Bull. no. 64. 
U.S. Dept. Agric. pp. 49. pis. 16. 1914. 

28. Pethybridge, G. H., and Bowers, G. E. H., Dry rot of the potato tuber. 
Econ. Proc. Roy. Dublin Soc. i : 547-558. pi. 48. 1908. 

29. PizziGONi, A., Cancrena secca ed unida delle patate. Nuovo Giov. Bot. 
Ital. 3:50-53- 1896. 

30. Reinke, J., and Bertholdt, G., Die Zersetzung der Kartoffel durch Pilze. 
Untersuch. Bot. Lab. Univ. Gottingen, Berlin. 1879. 

31. Rolfs, F. M., Potato failures. A preliminary report. Bull. no. 70. Coll. 
Agric. Exp. Sta. pp. 19. pis. 12. 1902. 

32. , Potato failures. A second report. Bull. no. 91. Coll. Agric. 

Exp. Sta. pp. ii. pis. 5. 1904. 

33. Sherbakoff, C. D., Fusaria of potatoes. Memoir. Cornell Univ. Agric. 
Exp. Sta. 6:90-270. pis. 7. 1915. 

34. Smith, E. F., Wilt disease of cotton, watermelon, and cowpea {Neocosmo- 
spora, n. gen.). Bull. U.S. Dept. Agric. Div. Veg. Phys. and Path. 17: 
1-26. pis. 10. 1899. 

35. Smith, E. F., and Swingle, D. B., Dry rot of potatoes due to Fusarium 
oxysponim. Bull. no. 55. Bur. PI. Ind. U.S. Dept. Agric. pp. 64. pis. 8. 
figs. 2. 1904. 

36- SoRAUER, Paul, Pflanzenkrankheiten. Berlin. 1886. 

37. Stewart, F. G., and Gloyer, W. O., The injurious effect of formaldehyde 
gas on potato tubers. Bull. no. 369. N.Y. Agric. Exp. Sta. 385-416. 
pis. 3. 1913. 

38. Wehmer, C, tJber die Ursache der sogenannten " Trockenf aule " der 
KartoffelknoUen. Ber. Deutsch. Bot. Gesells. 14:101-107.^^5. 3. 1896. 

39. , Ansteckungsversuche mit Fusarium Solani (Die Fusarium Faule). 

Centralbl. Bakt. u. Par. 3:727-743. pis. 10, 11. 1897. 

40. Wilcox, E, M., Link, Geo. K. K., and Pool, V. W., A dry rot of the Irish 
potato tuber. Research Bull. Neb. Agric. Exp. Sta. i: pp. 88. pis. 28. 

1913- 

41. WoLLENWEBER, H. W., Studies on the Fusarium problem. Phytopath- 
ology 3:24-50- pis. 5. fig. I. 1913. 

42. , Pilzparasitare Welke-Krankheiten der Kulturpflanzen. Ber. 

Deutsch. Bot. Gesells. 31:17-34. 1913. 



NOV 27 1916 • 



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